Antioxidant compositions and methods

ABSTRACT

Antioxidant compositions and methods are disclosed that are prepared by the extraction of natural antioxidants from plant substrates.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.726,540, filed Apr. 24, 1985, now abandoned.

BACKGROUND OF THE INVENTION

This invention is concerned with novel compositions and methods that maybe used for cosmetics, food preservation or therapeutic purposes.

The use of specific materials to inhibit or prevent oxidativedegradation of natural or synthetic materials is well known in the art.Many of the materials that have been utilized for the purpose have beeninsoluble in water and have been toxic to mammals at both high and lowlevels. Examples of these materials include BHA (butylated hydroxyanisole) BHT (butylated hydroxy toluene); propyl gallate; and alphatocopherol. It is known that natural antioxidants are widely distributedin plant tissues. Certain of these antioxidants have been obtained incrude form and have been shown to have an effect on commercial soybeanenzyme lipoxygenase (J. Food Science, V. 36 p 571/1971) Japanese patentapplication No. SHO 58-42686 discloses an alkali-organic solventextracting process that obtains an antioxidant from white pepper power.

In Medycyna Weterynaryjna 28:430-433, a procedure is disclosed forextracting dried hay or Urtica with boiling distilled water. The productwas used within 48 hours of its preparation as an antioxidant for fishmeal.

The applicants have discovered that antioxidants that may be obtained bywater extraction of plant tissues are absorbed percutaneously throughthe skin and exert an antioxidant effect on the outer and inner layersof the skin. These effects are advantageously obtained when theantioxidants is applied to the skin as a dispersion in a hydrophillic orhydrophobic base. The cosmetic result from the application of theantioxidant comprises a softening of the skin which is detectable bytouching with the fingertips. In addition, it has been found that theperoxide level of the skin is reduced by application of the antioxidant.These compositions may also be utilized for the preservation of food inplace of antioxidants such as BHT or BHA.

SUMMARY OF THE INVENTION

The invention comprises compositions and methods which relate to the useof a water soluble antioxidant, derived from plant tissues, which iscapable of being absorbed into mammalian skin where it reduces theperoxide level. The antioxidants may be used for food preservation andfor therapeutic purposes.

The plant tissues from which these water soluble antioxidants may beobtained are the leaves of Spinacia oleracea (spinach); Trifolium(clover); Medicago sativa (alfalfa); Zea mays (corn); Nicotiana tabacum(tobacco); Penicillaria; Allium (onion and garlic); Algae; and the like.Other suitable plants may also be utilized.

Accordingly it is a primary object of this invention to provide a novelantioxidant material.

It is an object of this invention to provide a cosmetic composition andmethods which may be used for application to the skin.

It is an object of this invention to provide a composition that may beabsorbed through the skin to provide an antioxidant effect.

It is also an object of this invention to provide compositions andmethods for therapeutic purposes.

It is also an object to provide compositions and methods for preventingoxidation in foods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart which shows the synergistic results of the use ofselected antioxidant fractions.

FIG. 2 shows a graphical comparison of the antioxidant effect of thecomposition of the invention with BHT.

FIG. 3 shows an infrared curve of the antioxidant fraction A of theinvention, isolated from spinach.

FIG. 4 shows an infrared curve of the antioxidant fraction B of theinvention, isolated from spinach.

FIG. 5 shows an infrared curve of the antioxidant fraction C of theinvention, isolated from spinach.

FIG. 6 shows an infrared curve of the antioxidant fraction C₁ of theinvention, isolated from spinach.

FIG. 7 shows an infrared curve of the antioxidant fraction A₁ of theinvention, isolated from spinach.

FIG. 8 shows an infrared curve of the antioxidant fraction A of theinvention, isolated from clover.

FIG. 9 shows an infrared curve of the antioxidant fraction B of theinvention, isolated from clover.

FIG. 10 shows an infrared curve of the antioxidant fraction C of theinvention, isolated from clover.

FIG. 11 shows an infrared curve of the antioxidant fraction A₁ of theinvention, isolated from clover.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides cosmetic compositions for application tothe skin. These compositions comprise a cosmetically acceptable carrierand an effective amount of a water soluble extract from plant tissuesuch as plant leaves and stems which is capable of being absorbedthrough the skin and lowering the peroxide level of the skin.

The plants that may be utilized as a source of the water soluble extractinclude the plant tissues of selected speices such as the stems andgreen leaves selected from the group consisting of Spinacia, Trifolium,Medicago, Zea, Penicillaria, Algae, and Nicotiana. Other plants may beutilized if an aqueous extract of the green leaves provide anantioxidant effect when it is applied to skin. The antioxidant effect isdetermined by the thiobarbituric acid (TBA) test. This test is describedin Food Res. V. 23, P 620 (1958). Generally the level of peroxide in theskin may be determined by assay of a sample of untreated skin which ispeeled from a test animal. A preweighed sample of from 10 to 50 mg ishomogenized in 0.2M phosphate buffer pH 6.5 and centrifuged. Thesupernatant is collected and the peroxide level is determined using theTBA test. A sample of skin from the same animal which has been treatedaccording to the invention is also peeled and the peroxide level isdetermined. A reduction in the peroxide level of about 35% when anantioxidant obtained from a plant is applied as 0.5% w/w dispersion in apetrolatum base is the criteria for determining if a given plant extractis useful according to the present invention.

The cosmetically acceptable carrier may be any liquid or semi-solid typeof material that is compatible with the plant extract and non-irritatingto the skin.

The water soluble antioxidant may be extracted from the plant materialusing a plant to water ratio of 0.5:100 to 1.0:5 (w/v as g/ml),preferably 2:1 (w/v as g/ml) after comminution of the plant material.The comminution may be carried out at temperatures of 4° C. to 100° C.,preferably at 25° C. using a blender, grinding apparatus or any type ofapparatus that will cause fragmentation of the cell walls. The extractedplant material is separated using filtration, centrifugation,decantation, froth flotation or other conventional method used forseparating a solid from a liquid.

The crude antioxidant may be used as obtained from the plant either indilute form or as an aqueous mixture or as a purified extract. Generallyit is preferred to separate the aqueous extracting medium from thedissolved antioxidant by evaporation or lyophilization of the liquidportion to provide a dry, water soluble antioxidant. The crude extractmay be purified using chromatographic techniques.

Generally, the powder is dissolved in water to form a 10 to 30% w/wsolution which is applied to the top of the column and is allowed tomove through the column. The various fractions are eluted using water aswashing medium and the various fractions are separately collected. Theindividual fractions may be further purified by a second chromatographicprocedure using a packing medium having a smaller pore size.

Sephadex G-25 may be utilized as a chromatographic column separationmedium to resolve the crude extract from spinach into a brown fraction(A), a yellow fraction (B) and an orange fraction (C). The orangefraction may be extracted with water and further separatedchromatographically using a Sephadex G-10 column. Sephadex G-25, mediumgrade, is dextran that has been cross-linked with epichlorohydrin andhas a pore size of 50-150-μm. Sephadex G-10 is dextran that has beencrosslinked with epichlorohydrin and has a pore size of 40-120 μm. Thinlayer chromatography is utilized to separate a yellow fraction from theorange fraction. The Sephadex materials are described in Gel FiltrationTheory and Practice, Pharmacia pp 1-64, which is incorporated byreference.

The applicants have isolated several different active antioxidantfractions which may be used separately or in combination. Several of thecombined fractions have been shown to have higher activity than thecrude fraction.

The relative amounts of the brown, orange and yellow fractions may bevaried to give optimum results. Generally any two fractions may be usedat weight ratios of 1:99 to 99:1 based on the total weight of thecombined fractions. If desired, more than two fractions may be combinedat any desired ratio.

For cosmetic use, the total amount of antioxidant that may be used mayvary from 0.005 to 5% by weight based on the total weight of theproduct. A preferred range is from 0.1 to 1%.

The nature of the cosmetic base material is not critical and anysuitable cosmetic cream or lotion may be utilized.

The antioxidant may be used in lipstick, face cream, body lotion,moisture creams, burn remedies containing local anesthetics such as 1%benzocaine and the like. The antioxidant has a protective effect againstdamage to the skin that is induced by UV light having a frequency in therange of 200-340 nm. Therefore, the antioxidant may be applied to theskin, to prevent damage caused by radiation from natural or artificialsources such as the sun, either alone or in combination with sunscreenagents such as PABA.

When foods are preserved with the compositions of the invention aneffective amount to prevent oxidation should be used. Generally from0.001%-1% or more preferably 0.005-0.1% by weight based on the totalweight of the foodstuff and antioxidants may be used depending on thefoodstuff and the type of oxidative activity that is to be inhibited.

Foods which contain fats or oils such as fatty acid esters or free fattyacids which are saturated or unsaturated may be preserved using thewater soluble antioxidant. The fatty acids are well known and are listedin Noller, Textbook of Organic Chemistry, 2nd Ed. pp 108-113 and 138-146(1958) which is incorporated by reference. Typical foods that containthese fats include soybean oil, corn oil, cottonseed oil, olive oil,butter, margarine, dairy products, ice cream, frozen vegetables, soups,fried foods and the like. In addition, foods which contain compoundsthat are susceptible to free radical oxidation may be protected with thecompounds of the invention. These include foods that contain flavones,carotenoids, tocopherols and the like.

It has also been found that both the crude and purified extracts arestable to high temperature, i.e., boiling water for 30 minutes and havegood stability for extended periods at ambient conditions. Toxicitystudies have been carried out using both crude and purified fractionsand no pathological changes have been detected when the materials havebeen administered by injection or orally.

The antioxidant has also been shown to be effective in inhibiting tumorssuch as fibrosarcoma induced by methylcholanthrene and skin cancer suchas squamous cell carcinoma which is induced bydimethylbenzoicanthracene, and 4B-phorbol 12-myristate-13-acetate. Thecompounds may be administered for this purpose at dosages of from 20-500mg/Kg of body weight orally, rectally or parenterally such as I.P. inmammals such as mice for inhibiting tumors. The invention also includespharmaceutical compositions that comprise the antioxidants of theinvention, together with an inert diluent or carrier. The compound maybe administered topically to prevent skin cancers such as melanoma. Thecompound may be applied in a cosmetically acceptable carrier at a levelof 0.005% to 5% by weight.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Leaves from Spinacia oleracea were homogenized with H₂ O at 25° C. at aratio of 2:1 (w/v g/ml) in a Waring blender for 5 min. The resultinghomogenate was filtered through cheesecloth and then centrifuged at15000 X g for 10 min. The supernatant was collected and lyophilized.

Isolation and purification

THe isolation and purification of antioxidant fractions for the crudehomogenate preparation was achieved through gel filtration followed bypreparative TLC or HPLC. One gram of the lyophilized powder of the crudehomogenate was dissolved in 5 ml H₂ O and after centrifugation at 20,000X g for 10 min, the supernatant was applied to a Sephadex G-25 column(40 cm×2.5 cm) equilibrated and eluted with H₂ O. Fractions of 5 ml werecollected and each was assayed for antioxidant activity. The activefractions (A, B and C) were pooled (fraction A has a brown colour,B-yellow and C-orange) and lyophilized. Fraction C was further purified.The lyophilized material of fraction C was dissolved in H₂ O to form a20% solution (w/v), centrifuged at 20,000 X g for 10 min and thesupernatant was chromatographed on a Sephadex G-10 column (40 cm×2.5 cm)equilibrated with H₂ O. Fractions C₁ and C₂ were collected separatelyand lyophilized as before. Lyophilized fraction C.sub. 1 was dissolvedin a minimum amount of H₂ O, applied to 0.2 mm silica gel plates(DC-Karten SIF, Riedel-Dollaen Ag, sleeze-Hanover) and developed in H₂O/ethanol (30/60, v/v). The active fraction was identified by its weak(pale) yellow color and was extracted from the silica gel plate with H₂O and lyophillized.

A further purification was carried out using DEAE cellulose (smallsize). The fraction identified hereinabove as A was dissolved in waterand passed through a 5 cm×1 cm column packed with DEAE cellulose (smallsize) that had been equilibrated with water that was acidified to a pHof 5-6 with 0.2N HCl. Thereafter the column was washed first with about50 ml of water and thereafter with 50 ml of aqueous HCl pH 4. The columnwas eluted with a solution of HCl pH 2.0 and the eluted material wasrecovered as a powder by vacuum evaporation and was identified asfraction A₁. The powder was dissolved in water at a concentration of 20ug/ml and passed through a high pressure liquid chromatography silica 60column (250 mm×4 mm) with a 90:10 solution of water:acetonitrile appliedat a rate of 0.5 ml/minute. A fraction was obtained that had a retentionfraction at 5.4 nanometers (UV absorption).

Fraction A₁ had the infrared curve of FIG. 7 and an elemental analysisof C₈ H₁₀ O₆ N₁ ; found C 42.02%; H 4.80%; N 6.38%; O 40.09%.

An alternative column packing may be Ecteola, a commecially availablecondensation product of cellulose with epichlorohydrin andtriethanolamine having a capacity of 0.3 to 0.4 meg/g and a particlesize of 0.05-0.2 mm.

EXAMPLE 1

From the crude extract of the plant material three antioxidant activefractions (A, B and C) were obtained following the first step ofpurification. Fraction C was further purified on a column packed withSephadex G-10 and two other active fractions were obtained by elutionwith water (C₁ -dark brown and C₂ -yellow orange). Fraction C₁ wasfinally purified using HPLC. In studying the antioxidant activity of thecrude plant extract and the isolated fractions, both the inhibition oflinoleate oxidation by lipoxygenase and the inhibition of auto-oxidationof peroxides were used as criteria for antioxidant activity.

The antioxidant fractions exhibited synergistic activity. The synergismobtained with the natural isolated antioxidants is described in FIG. 1,which depicts the percentage inhibition on lipid oxidation of 1 mg eachof single purified antioxidant fractions as well as the analogouspercentage inhibition using combinations of 0.5 mg each of two suchfractions. By way of example, it may be seen that this synergismincreased the potency produced by the compounds from 167% (B+C₂) up to250% (A+B), without increasing the total antioxidant content.

Since lipid peroxidation catalyzed by hemeproteins is a basicdeteriorative and pathological reaction, the effectiveness of theisolated fractions to prevent such peroxidation was followed. It wasfound that the isolated fractions prevent such peroxidation induced byhemoglobin, cytochrome C and myoglobin in a similar way to theinhibition of lipoxygenase induced oxidation.

The purified antioxidant fractions retained their antioxidativeactivities for months without any loss when kept at room temperature.Moreover, boiling the purified antioxidants for up to 30 minutes did notreduce their antioxidant capacity.

The following infrared data was obtained from the spinach-derivedfractions:

A: (see FIG. 3) broad band at 3400 cm.⁻¹, strong bands at 1050 and 1650cm.⁻¹, weak bands at 1250 and 1430 cm.⁻¹.

B: (see FIG. 4) broad bands at 3400, 1640 and 1080 cm.⁻¹, additionalbands at 1420, 1300 and 810 cm.⁻¹.

C: (see FIG. 5) broad bands at 3400 and 1600 cm.⁻¹, strong band at 1390cm.⁻¹, additional bands at 1070 and 820 cm.⁻¹.

C₁ : (see FIG. 6) broad band at 3300 cm.⁻¹, strong band at 1620 cm.⁻¹,additional bands at 1390, 1320, 1080 and 770 cm.⁻¹.

A₁ : (see FIG. 7) broad band at 3300-3400 cm.⁻¹, strong band at 1650cm.⁻¹, addl. bands at 1730, 1540, 1250 and 1080 cm.⁻¹, weak bands at2920, 1400 and 1150 cm.⁻¹.

EXAMPLE 2

Samples of creams and appropriate controls were applied to mice or ratskin for a fixed period. The application was done once a day.Experiments were terminated by killing the animals, peeling the skin andfreezing it in liquid nitrogen. Samples of the frozen skin werehomogenized in 0.2M phosphate buffer, pH 6.5. After centrifugation thesupernatant was collected and analyzed for the peroxide value using theTBA (Thiobarbituric acid) test as described by Sinnhuber, et al. FoodRes. V. 23, p 620 (1958).

Tests with Newborn Rats:

In these experiments newborn rats (hairless) were tested. It isgenerally considered that the penetration through the skin of newbornrats is better than in adult rats. The advantage of using these rats wasthat at this early stage they had not yet developed any fur.

TEST NO. 1

In this experiment the control group was treated with Vaseline only,while the test group was treated with Vaseline containing a C₁ fractionin Vaseline. A relatively larger amount of the C₁ fraction was taken andsuspended in the Vaseline. The test was run for 12 days and the resultsare presented in Table 1.

                  TABLE 1                                                         ______________________________________                                                  TBA          Level of     P value*                                  GROUP     O.D..sub.532 /1 g. tissue                                                                  Peroxidation (n = 3)                                   ______________________________________                                        Control   0.295        100%         0.002                                     +C.sub.1 (0.5%)                                                                         0.188        64%          0.002                                     ______________________________________                                         *standard deviation                                                      

It is clearly demonstrated that the C₁ penetrates the skin of newbornrats and consequently reduces the level of peroxides in the skin. Sinceperoxides and free radicals involved in their formation and breakdownconstitute one of the main routes leading toward aging, the activity ofthis unique antioxidant can be considered as an anti-aging factor.

TEST NO. 2

In this experiment the antioxidant was dissolved in Oil of Olay obtainedin Israel (excellent solubility) and experiments similar to thatdescribed in No. 1 was performed. The data are presented in Table 2.

                  TABLE 2                                                         ______________________________________                                                   TBA O.D..sub.532 /                                                                        Level of     P value*                                  GROUP      1 g. tissue Peroxidation (n = 3)                                   ______________________________________                                        Control                                                                       (no treatment)                                                                           0.295       100%         0.002                                     Control                                                                       (Oil of Olay)                                                                            0.230       78%          0.005                                     C.sub.1 (0.15%)                                                                          0.200       68%          0.011                                     C.sub.1 (1.5%)                                                                           0.191       65%          0.010                                     ______________________________________                                         *standard deviation                                                      

As in test No. 1, the Antioxidant significantly reduced the level ofperoxides in the skin. It is interesting to point out that in newbornrats, Oil of Olay without the antioxidants also reduced the level ofperoxides. This may be attributed to the commercial antioxidants thatwere present in Oil of Olay that was used. It is possible that innewborn skin, due to its relatively high permeability, small amounts ofthese antioxidants can also penetrate the skin. However, in adult miceor rats, as will be shown later, Oil of Olay did not reduce that levelof peroxides in the skin. On the contrary, in general, a small increasein peroxide level was detected (which perhaps may be attributed totraces of metals in the cream).

EXAMPLE 3

In these experiments adult mice (2 months old) were treated as describedin Example 2. The grown mice were shaved before applying the creams tothe skin.

In this experiment the antioxidant was dissolved in Oil of Olay. Micewere sacrificed after 21 days. The data are presented in Table 3.

                  TABLE 3                                                         ______________________________________                                                   TBA O.D..sub.532 /                                                                        Level of     P value*                                  GROUP      1 g. tissue Peroxidation (n = 3)                                   ______________________________________                                        Control                                                                       (without cream)                                                                          0.338       100%         0.019                                     Oil of Olay                                                                              0.400       118%         0.026                                     C.sub.l (0.3%)                                                                           0.240       71%          0.002                                     ______________________________________                                         *standard deviation                                                      

It seems that in grown mice the Oil of Olay slightly increases the levelof peroxides while addition of the antioxidant at a concentration of0.3% significantly reduced these peroxides, thus indicating that evenwith grown mice the antioxidant penetrates the skin. We would like topoint out that in similar experiments when we tried the effect of 0.1%BHT, BHA and alpha tocopherol dissolved in Oil of Olay on the level ofperoxides in the skin no reduction of the peroxides was observed.

EXAMPLE 4

A new model for studying aging was developed. The new model involves theexposure of adult shaved mice to a UV lamp (sun Lamp 300 W) for a shortperiod. As a result the aging processes as expressed by the level ofperoxidation are stimulated and the effect of the natural antioxidantwas studied. Using this new technique the optimal antioxidant dose forthe inhibition of aging was determined.

In this experiment a crude preparation of antioxidant (and not the finalpurified antioxidant) was used.

Adult mice were shaved and individuals were exposed to the UV light(Philips HP 3115) (with or without antioxidant) for a short period of 1minute for two days (two exposures in total). On the third day they weresacrificed and the level of peroxidation in the skin was determined bythe TBA (thiobarbituric acid) test.

Controls without exposure to the UV light were also included.Antioxidant was dissolved in Oil of Olay. Data are presented in Table 4.

                  TABLE 4                                                         ______________________________________                                        EFFECT OF ANTIOXIDANT DOSE ON AGING                                           (EACH GROUP CONTAINED 7 INDIVIDUALS)                                                       TBA O.D..sub.532 /                                                                        Level of   P value*                                  GROUP        1 g. tissue Peroxidation                                                                             (n = 3)                                   ______________________________________                                        1.  No radiation 0.147       16.7%    0.010                                   2.  Radiation +                                                                   Oil of Olay  0.880       100%     0.027                                   3.  Radiation + 0.3%                                                              Antioxidant in                                                                Oil of Olay  0.740       84%      0.006                                   4.  Radiation + 0.4%                                                              Antioxidant in                                                                Oil of Olay  0.680       77%      0.020                                   5.  Radiation + 0.5%                                                              Antioxidant in                                                                Oil of Olay  0.680       77%      0.011                                   6.  Radiation + 1%                                                                Antioxidant in                                                                Oil of Olay  0.700       79%      0.006                                   ______________________________________                                         *standard deviation                                                           The optimal dose of crude antioxidant to be used is 0.3-0.4%.            

EXAMPLE 5

Samples of human skin were obtained from a Plastic Surgery Department ofa Hospital. These samples were placed in saline solution immediatelyafter their removal from the patients.

The skin samples were exposed to UV rays (Phillips Sun Lamps) for 5 minintervals three successive times with a 5 min rest period between eachexposure. The distance between the lamp and the tissue was 12 cm. Theskin samples were stored for three days at 4° C. after which time theywere peeled and homogenized. 20-30 mg of peeled tissue were assayed forperoxide level using the spectrophotometric TBA test.

The results clearly demonstrate that the peroxide level (aging) of theskin tissue was raised due to exposure to UV rays. Skin treated with thenatural antioxidant and exposed to UV rays for the same period of timeshowed a peroxide level similar to the untreated control.

These results are shown in Table 5.

                  TABLE 5                                                         ______________________________________                                                       TBA (O.D..sub.532 /0.1                                                                      Level of                                         SAMPLE         gram tissue)  Peroxidation                                     ______________________________________                                        Unexposed      0.050         62.5                                             Exposed        0.080         100                                              Exposed + Oil of Olay                                                                        0.100         125                                              Exposed + Oil of Olay                                                         + (A + B + C)  0.050         62.5                                             ______________________________________                                    

The experiments run on human skin indicate the following:

(a) The antioxidant penetrates the skin;

(b) The antioxidant significantly reduces the level of peroxides.

It was noted that when a mixture of fraction A, B and C were used, aneffective antioxidant result was observed in the skin.

EXAMPLE 6

The crude extract was tested in vivo for its effect on the immuneresponse system in experimental mice. In these experiments, male Balb-Cmice were injected intraperitoneally with 1 mg of the crude extract fromSpinacia oleracea per 0.2 ml of phosphate buffer solution (PBS) peranimal. Animals were sacrificed one, three and seven days afterinjection and their spleens removed. Spleen cells (10⁷ cell/ml enrichedRPMI) were cultured for 24 hours in the presence of CON A (concavalin-A)2 μg/ml and the supernatants thus obtained were tested both for lL-2 and(interlukine-2) and CSF (colony stimulating factor). No significantdifferences were found between controls (i.e., animals receiving notreatment) and experimental animals in their ability to produce lL-2 aswell as CSF indicating that the antioxidant has no adverse effect on theimmune system. In addition, no pathological findings were observed ininjected animals.

Additional testing determined that a single dose of 25 mg/mouse IP maybe tolerated and that the LD₅₀ is in the range of 1400-1500 mg/kg formice.

EXAMPLE 7

The C₁ fraction was dissolved in (PBS) (50 mg of C₁ fraction in 10 mlPBS) 0.2 ml was injected IP into each mouse twice weekly. The C₁fraction was administered orally in an aqueous solution (1 mg/ml) andthe mice were allowed to drink the solution from a calibrated bottle toenable measurement of the quantity of the C₁ fraction consumed by eachindividual mouse. Each mouse was injected with 0.6 mg ofmethylcholantrene, a known inducer of fibrosacoma. Test series A and Bwere carried out as follows:

    ______________________________________                                        Tumor Appearance                                                              (Animal with tumor/animals in group)                                          Weeks After                                                                   Innoculation                                                                  with MC.  Groups treated with                                                 Methylchol-                                                                             Control Oral Anti-                                                                            Groups treated with                                 anthrene  oxidants        I.P. Antioxidants                                   ______________________________________                                        TEST A                                                                        5 weeks   4/20      1/10      1/10                                            6 weeks   9/20      1/10      1/10                                            7 weeks   14/20     3/10      2/10                                            8 weeks   16/20     3/10      2/10                                            9 weeks   18/20     4/10      2/10                                            TEST B                                                                        7 weeks   1/10      0/8       0/9                                             8 weeks   3/10      0/8       0/9                                             9 weeks   4/10      0/8       0/9                                             10 weeks  4/10      0/8       0/9                                             11 weeks  6/10      1/8       0/9                                             12 weeks  7/10      1/8       0/9                                             13 weeks  7/10      2/8       1/9                                             ______________________________________                                    

At week 13 (Test B) (after as many as 25-29 injections) one mouse fromeach group was sacrificed and observed for gross internal changes (i.e.,lymph nodes, spleen, liver, kidney, heart and lung, etc.); nosignificant changes and no pathological damage was observed. Thisdemonstrated that even a prolonged treatment with the C₁ fraction bydifferent routes of administration did not cause any damage to thetreated mice.

The in vivo experiments demonstrated that IP or oral administration withC₁ is effective in delaying the appearance and reducing the frequency ofmethylcholanthrene induced tumors.

EXAMPLE 8

Skin tests on human volunteers using a 0.3% w/w dispersion of the crudeextract in Oil of Olay have resulted in subjective improvement in thetexture of the skin with no adverse effects in any test subjects.

EXAMPLE 9

This example illustrates various compositions that may be used in thepractice of the invention.

    ______________________________________                                        Lotion                                                                        Antioxidant (Example 7)                                                                              1.0      g                                             Base*                  99.0     g                                                                    100.0    g                                             *stearic acid          1.4      g                                             triethanolmine         0.6      g                                             glyceryl monostearate  4.0      g                                             lanolin, hydrous       1.0      g                                             cetyl alcohol          0.4      g                                             mineral oil            2.0      g                                             methylparahydroxybenzoate                                                                            0.1      g                                             distilled water        90.5     g                                             perfume                g.s. 100.0                                                                             g                                             CREAM                                                                         antioxidant            1.0      g                                             cetyl alcohol          6.4      g                                             stearyl alcohol        7.4      g                                             isopropyl myristate    2.0      g                                             sodium lauryl sulfate  1.4      g                                             white petrolatum       27.6     g                                             propylene glycol       9.2      g                                             water, to make         100.0    g                                             ______________________________________                                    

EXAMPLE 10

The crude antioxidant (A, B and C) was added to linoleic acid to form amixture containing 20 ml of 7.5×10⁻³ of linoleic acid in 0.2M aqueoussodium phosphate buffer (pH 6.5), containing 0.25% Tween 20 and 1 mg ofthe crude antioxidant. Controls were run which contained the buffer andTween 20 but no antioxidant as well as a sample of linoleic acid with 1mg of BHT and the same dispersant system. The mixture was kept at 30°and the optical density was determined using the ferric thiocyanatemethod described by Koch et al. in Arch. Biochem. Biophys. Vol. 78, p165 (1959). The test results on FIG. 2 show that the antioxidant of theinvention is more effective than BHT in preventing oxidation of thelinoleic acid.

EXAMPLE 11

A similar procedure to that described for spinach, was applied toisolate antioxidant materials from Trifolium alexandrinum. The crudeextract was separated on Sephadex G-25 to give fractions A, B and C.Fraction A was purified on Ecteola to give fraction A₁. Fraction C wasresolved on Sephadex G-10 to give fractions C₁ and C₂. Fraction C₁ wasfurther resolved by dissolving in a minimum amount of water, applying to0.2 mm. silica gel plates and developing in 30:60 v/v H₂ O-ethanol, togive fractions labelled TLC-1, -2 and -3.

The following infrared data was obtained:

A: (see FIG. 8).

B: (see FIG. 9) strong and broad bands at 3300, 1560 and 1130 cm.⁻¹,medium band at 1400 cm.⁻¹, weak bands at 1350 and 1430 cm.⁻¹.

C: (see FIG. 10) broad band at 1370 cm.⁻¹, strong bands at 1600, 1380and 1150 cm.⁻¹.

A₁ : (see FIG. 11).

Certain of the foregoing fractions (0.2 mg in each case) derived fromclover were tested as antioxidant in a system which contained linoleicacid as substrate and the enzyme lipoxygenase as catalyst. Oxygenabsorption was followed using an oxygen monitor according to Grossmanand Zakut, in Methods of Biochemical Analysis (D. Glick, Ed.) 25: 303-29(1979). The following results were obtained.

    ______________________________________                                        Inhibition of Lipid Peroxidation                                              by Antioxidants from Clover                                                   Fraction       % Inhibiton                                                    ______________________________________                                        crude extract  20                                                             A              9                                                              B              16                                                             C              30                                                             TLC-1          42                                                             TLC-3          46                                                             ______________________________________                                    

EXAMPLE 12

A number of algae samples were homogenized with distilled water and anextract was prepared according to the technique described above forSpinacia oleracea. The crude homogenate was centrifuged and thesupernatant was collected and was dried by lyophilization. The driedcrude extracts were tested as antioxidants in a system which containedlinoleic acid as a substrate and the enzyme lipoxygenase as catalyst.Oxygen absorption was followed using an oxygen monitor according toGrossman and Zakut in Methods of Biochemical Analysis (D. Glick, Ed.)25, 303-329 (1979). The following results were obtained (2.5 mg of crudeextract was used):

    ______________________________________                                        Inhibition of Lipid Peroxidation                                              by Antioxidants from Algae                                                    Algae          % Inhibition                                                   ______________________________________                                        Spirulina      30                                                             Nicractinium   27                                                             Synichococcus  30                                                             Navicola       42                                                             Euglena        35                                                             Red            35                                                             ______________________________________                                    

While the invention has been described above with respect to itspresently preferred embodiments, it will be apparent to those skilled inthe art that many variations and modifications may be made. Theinvention is accordingly not to be construed as restricted to theillustrated embodiments, rather its scope will be defined in the claimswhich follow.

We claim:
 1. A composition for application to the skin, said compositioncomprising a cosmetically acceptable carrier and cosmetically effectiveamounts of a water soluble extract from plant tissue, said extract andat least two distinct fractions chromatographically separable therefrombeing capable of being absorbed through the skin and lowering theperoxide level of the skin, said plant being selected from the groupconsisting of Spinacia, Trifolium, Medicago, Nicotiana, Penicillaria,Zea, and Allium.
 2. A composition as defined in claim 1 wherein thecosmetically acceptable carrier is a hydrophillic cream.
 3. Acomposition as defined in claim 1 wherein the cosmetically acceptablecarrier is a hydrophillic lotion.
 4. A composition as defined in claim 1wherein the cosmetically acceptable carrier is a hydrophobic cream.
 5. Acomposition as defined in claim 1 wherein the cosmetically acceptablecarrier is a hydrophobic lotion.
 6. A composition as defined in claim 1wherein said water soluble extract from plant tissue is an orangefraction that is chromatographically separated from the supernatantobtained by aqueous extraction of said plant tissue using dextran thatwas cross-linked with epichlorohydrin and having a pore size of 50-150μm as the packing for the chromatographic column.
 7. A composition asdefined in claim 1, wherein said water soluble extract from plant tissueis a brown fraction that is chromatographically separated from thesupernatant obtained by aqueous extraction of said plant tissue usingdextran that was cross-linked with epichlorohydrin and having a poresize of 40-120 μm as the packing for the chromatographic column.
 8. Acomposition as defined in claim 1, wherein said water soluble extractfrom plant tissue is a yellow fraction that is chromatographicallyseparated from the supernatant obtained by aqueous extraction of saidplant tissue being dextran that has been cross-linked withepichlorohydrin and having a pore size of 50-150 μm as the packing forthe chromatographic column.
 9. A composition as defined in claim 1,wherein said water soluble extract comprises a mixture of two or more ofthe orange, yellow and brown fractions that are chromatographicallyseparated from the supernatant obtained by aqueous extraction of saidplant tissue being dextran that has been cross-linked withepichlorohydrin and having a pore size of 40-120 μm as the packing forthe chromatographic column.
 10. A method of cosmetically enhancing thetexture of the skin which comprises applying to the skin a compositioncomprising a cosmetically acceptable carrier and cosmetically effectiveamounts of a water soluble extract from plant tissue, said extract andat least two distinct fractions chromatographically separable therefrombeing capable of being absorbed through the skin and lowering theperoxide level of the skin and said plant being selected from the groupconsisting of Spinacia, Trifolium, Medicago, Nicotiana, Penicillaria,Zea, and Allium.
 11. A method of cosmetically enhancing the texture ofthe skin which comprises applying to the skin the composition of claim2.
 12. A method of cosmetically enhancing the texture of the skin whichcomprises applying to the skin the composition of claim
 3. 13. A methodof cosmetically enhancing the texture of the skin which comprisesapplying to the skin the composition of claim
 4. 14. A method ofcosmetically enhancing the texture of the skin which comprises applyingto the skin the composition of claim
 5. 15. A method of cosmeticallyenhancing the texture of the skin which comprises applying to the skinthe composition of claim
 6. 16. A method of cosmetically enhancing thetexture of the skin which comprises applying to the skin the compositionof claim
 7. 17. A method of cosmetically enhancing the texture of theskin which comprises applying to the skin the composition of claim 8.18. A method of cosmetically enhancing the texture of the skin whichcomprises applying to the skin the composition of claim
 9. 19. Anantioxidant composition of matter having the infrared curve of FIG. 7.20. An antioxidant composition of matter having the infrared curve ofFIG.
 3. 21. An antioxidant composition of matter having the infraredcurve of FIG.
 4. 22. An antioxidant composition of matter having theinfrared curve of FIG.
 5. 23. An antioxidant composition of matterhaving the infrared curve of FIG.
 6. 24. An antioxidant materialselected from the group consisting of a water soluble extract preparedfrom plant tissue and distinct fractions separable from said extract bychromatography, said material and at least two of said fractions beingcapable of being absorbed through the skin and lowering the peroxidelevel of the skin and said plant being selected from the groupconsisting of Spinacia, Trifolium, Medicago, Nicotiana, Zea,Penicillaria and Allium.
 25. An antioxidant material according to claim24, wherein the plant tissue is constituted by at least one memberselected from the group consisting of fresh leaves and stems.
 26. Anantioxidant material according to claim 24, wherein said extract ischromatographically separable on dextran which has been cross-linkedwith epichlorohydrin and has a pore size of 50-150 μm, into fractionswhich are colored brown(A), yellow(B) and orange(C), and of whichfraction A is chromatographically purifiable on a substance selectedfrom the group consisting of (i) a condensation product of cellulosewith epichlorohydrin and triethanolamine having a capacity of 0.3 to 0.4meq./g. and a particle size 0.05-0.2 mm., and (ii) dextran which hasbeen cross-linked with epichlorohydrin and has a pore size of 40-120 μm,to give a fraction (A₁) having an infrared spectrum with substantiallythe following features, namely, broad band at 3300-3400 cm.⁻¹, strongband at 1650 cm.⁻¹, additional bands at 1730, 1540, 1250 and 1080 cm.⁻¹,weak bands at 2920, 1400 and 1150 cm.⁻¹, and of which fractions,fraction C is chromatographically separable on dextran which has beencross-linked with epichlorohydrin and has a pore size of 40-120 μm, intofractions colored dark brown(C₁) and yellow orange(C₂).
 27. Anantioxidant material which comprises at least one substance selectedfrom the group consisting of fractions A, A₁, B, C₁ and C₂, as definedin claim
 26. 28. An antioxidant material which comprises anantioxidatively synergistic combination of fractions A, A₁, B, C₁ andC₂, as defined in claim
 26. 29. An antioxidant material, which isobtainable by extraction of plant tissue with water and subsequentfractionation by chromatographic methods, and having an infra-redspectrum with the features of a member selected from the groupconsisting of (i), (ii), (iii), (iv), (v), (vi) and (vii), namely:(i)broad band at 3400 cm.⁻¹, strong bands at 1050 and 1650 cm.⁻¹ weak bandsat 1250 and 1430 cm.⁻¹ ; (ii) broad bands at 3400, 1640 and 1080 cm.⁻¹additional bands at 1420, 1300 and 810 cm.⁻¹ ; (iii) broad band at 3300cm.⁻¹, strong band at 1390 cm.⁻¹, additional bands at 1070 and 820 cm.⁻¹(iv) broad band at 3300 cm.⁻¹, strong band at 1620 cm.⁻¹ additionalbands at 1390, 1320, 1080 and 770 cm.⁻¹ ; (v) broad band at 3300-3400cm.⁻¹, strong band at 1650 cm.⁻¹, addl. bands at 1730, 1540, 1250 and1080 cm.⁻¹, weak bands at 2920, 1400 and 1150 cm.⁻¹ ; (vi) broad band at3430 cm.⁻¹, strong bands at 1600, 1380 and 1150 cm.⁻¹. (vii) broad bandat 3430 cm.⁻¹, strong bands at 1600, 1380 and 1150 cm.⁻¹.
 30. A processaccording to claim 24, wherein said extracting step is carried out aftera preliminary step of comminuting said plant tissue.
 31. A processaccording to claim 24, wherein said preliminary step is effected at atemperature within the range of from about 4° to about 100° C.
 32. Aprocess according to claim 31, wherein said temperature is about 25° C.33. A process according to claim 24, wherein said fractionationcomprises chromatographically separating said extract on dextran whichhas been cross-linked with epichlorohydrin and has a pore size of 50-150μm, into fractions which are colored brown(A), yellow(B) and orange(C),and optionally effecting at least one of the following chromatographicpurifications on said fractions, namely:chromatographically purifyingfraction A on a condensation product of cellulose with epichlorohydrinand triethanolamine having a capacity of 0.3 to 0.4 meq./g. and aparticle size 0.05-0.2 m.m., or on dextran which has been cross-linkedwith epichlorohydrin and has a pore size of 40-120 μm, to give afraction(A₁) having an infrared spectrum with substantially thefollowing features, namely, broad band at 3300-3400 cm.⁻¹, strong bandat 1650 cm.⁻¹, additional bands at 1730, 1540, 1250 and 1080 cm.⁻¹, weakbands at 2920, 1400 and 1150 cm.⁻¹ ; chromatographically separatingfraction C on dextran which has been cross-linked with epichlorohydrinand has a pore size of 40-120 μm, into fractions colored dark brown(C₁)and yellow orange(C₂).
 34. A composition of matter for therapeutic orcosmetic use, which comprises a stable antioxidant material as definedin claim 24, together with an inert diluent or carrier.
 35. Acomposition according to claim 34, wherein said composition containsfrom about 0.005 to about 5% by weight of said antioxidant material,based upon the total weight of the composition.
 36. A compositionaccording to claim 34, wherein said composition is in the form of amember selected from the group consisting of a hydrophilic cream, ahydrophilic lotion, a hydrophobic cream and a hydrophobic lotion.
 37. Acomposition according to claim 36, wherein the inert diluent or carrieris adapted for oral, rectal or parenteral administration.
 38. A methodfor cosmetically enhancing the texture of the skin, which methodcomprises applying to the skin a composition which comprises aantioxidant material as defined in claim 24, together with an inertdiluent or carrier which is adapted for application to the skin.
 39. Amethod for cosmetically enhancing the texture of the skin, which methodcomprises applying to the skin a composition which comprises aantioxidant material as defined in claim 25, together with an inertdiluent or carrier which is adapted for application to the skin.
 40. Amethod for cosmetically enhancing the texture of the skin, which methodcomprises applying to the skin a composition which comprises aantioxidant material as defined in claim 26 together with an inertdiluent or carrier which is adapted for application to the skin.
 41. Amethod for cosmetically enhancing the texture of the skin, which methodcomprises applying to the skin a composition which comprises aantioxidant material as defined in claim 27, together with an inertdiluent or carrier which is adapted for application to the skin.
 42. Amethod for cosmetically enhancing the texture of the skin, which methodcomprises applying to the skin a composition which comprises aantioxidant material as defined in claim 28, together with an inertdiluent or carrier which is adapted for application to the skin.
 43. Amethod for lowering the peroxide level of the skin which comprisesapplying to the skin an effective amount of the composition according toclaim
 34. 44. A composition for providing an antioxidant effect to theskin, said composition comprising a cosmetically acceptable carrier andan cosmetically effective amount of a water soluble extract from thefresh leaves and stems of a plant selected from the group consisting ofSpinacia, Trifolium, Medicago, Nicotiana, Zea, Penicillaria and Allium,wherein said water soluble extract has been chromatographicallyseparated from the supernatant obtained by aqueous extraction of saidfresh leaves or stems.
 45. A composition as defined in claim 44, whereinthe water soluble extract is obtained by a process which consistsessentially of aqueous extraction of said stems and leaves by anextractant that consists essentially of water.
 46. A composition asdefined in claim 45, wherein the process is carried out at a temperatureof from 4°-100° C.
 47. A composition as defined in claim 46, wherein theprocess is carried out at the ambient temperature.
 48. A composition asdefined in claim 45, wherein said water soluble extract from planttissue is a brown fraction that has been chromatographically separatedfrom the supernatant obtained by aqueous extraction of said plant tissueusing dextran that was cross-linked with epichlorohydrin and having apore size of 40-120 μm as the packing for the chromatographic column.49. A composition as defined in claim 45, wherein said water solubleextract from plant tissue is a yellow fraction that has beenchromatographically separated from the supernatant obtained by aqueousextraction of said plant tissue using dextran that has been cross-linkedwith epichlorohydrin and having a pore size of 50-150 μm as the packingfor the chromatographic column.
 50. A composition as defined in claim45, wherein said water soluble extract comprises a mixture of two ormore of the orange, yellow and brown fractions that has beenchromatographically separated from the supernatant obtained by aqueousextraction of said plant tissue using dextran that has been cross-linkedwith epichlorohydrin and having a pore size of 40-120 μm as the packingfor the chromatographic column.
 51. A composition as defined in claim45, wherein said plant is selected from Spinacia.
 52. A composition asdefined in claim 45, wherein said plant is selected from Trifollium. 53.A composition as defined in claim 45, wherein said plant is selectedfrom Medicago.
 54. A composition as defined in claim 45, wherein saidplant is selected from Nicotiana.
 55. A composition as defined in claim48, wherein said plant material is selected from Allium.
 56. Acomposition as defined in claim 48, wherein said plant material isselected from Penicillaria.
 57. A method of cosmetically enhancing thetexture of the skin, which comprises applying to the skin a compositionas defined in claim
 44. 58. A method of cosmetically enhancing thetexture of the skin, which comprises applying to the skin a compositionas defined in claim
 45. 59. A method of lowering the peroxide level ofthe skin, which comprises applying to the skin an amount of thecomposition of claim 44, which will lower the peroxide content of theskin.
 60. A method of lowering the peroxide level of the skin, whichcomprises applying to the skin an amount of the composition of claim 45,which will lower the peroxide content of the skin.
 61. A composition asdefined in claim 45, wherein said antioxidant is stable to boiling waterfor 30 minutes.